DESCRIPTION [Verbatim from application]: The overall goal of this study is to understand how leukocytes respond to external stimuli and migrate to sites of infection and inflammation. Previous efforts have been focused on understanding the role of changes in intracellular free calcium [Ca2+i], in regulating adhesive interactions and the cytoskeleton. It was found that transient increases in [Ca2+i] are required for neutrophils to dissociate from vitronectin and fibronectin. The [Ca2+i]-sensitive binding to these matrix proteins is via alpha v beta 3 and alpha 5 beta 1 integrins, respectively. Under normal conditions, it was found that both of these integrins are distributed on the adherant membrane with a gradient that is higher at the front of the cell. These integrins are also in endocytic vesicles. When [Ca2+i] transients are blocked, the integrins are found at the rear of the cells on the adherant membrane, and endocytic vesicles do not contain the integrins. Based on these and other data it was proposed that [Ca2+i] transients are required to release the alpha v beta 3 and alpha 5 beta 1 integrins from tight binding and that after release they are internalized and recycled toward the front of a migrating cell. One aim of the proposed research is to investigate the oriented recycling in migrating neutrophils. Digital fluorescence microscopy, confocal microscopy, and electron microscopy will be used to examine the endocytic recycling pathways in neutrophils, and the passage of integrins through these pathways will be examined in detail. The role of microtubule-based vesicle motors will be examined by disruption of dynein motor function in neutrophils and neutrophil-like HL-60 cells by overexpression and/or cytoplasmic delivery of p50-dynamitin. Myosin II is a major cytoskeletal protein that is activated by increases in [Ca2+i]. The role of myosin II in neutrophil migration on 2D substrates and through natural 3D matrices will be examined. Using antibodies to myosin II and affinity purified antibodies to the phosphorylated form of myosin light chain, the distribution of myosin II and its activation under various conditions will be determined by immunofluorescence. Myosin II function will be inhibited by delivery of inhibitory peptides to the cytoplasm of migrating cells. Finally, the role of myosin and of oriented recycling will be examined in neutrophils migrating through endothelial cell monolayers and through natural biological matrices, which resemble the physiological sites of neutrophil function.